A high-throughput platform for screening milligram quantities of plant biomass for lignocellulose digestibility.

The development of a viable lignocellulosic ethanol industry requires multiple improvements in the process of converting biomass to ethanol. A key step is the improvement of the plants that are to be used as biomass feedstocks. To facilitate the identification and evaluation of feedstock plants, it would be useful to have a method to screen large numbers of individual plants for enhanced digestibility in response to combinations of specific pretreatments and enzymes. This paper describes a high-throughput digestibility platform (HTDP) for screening collections of germplasm for improved digestibility, which was developed under the auspices of the Department of Energy-Great Lakes Bioenergy Research Center (DOE-GLBRC). A key component of this platform is a custom-designed workstation that can grind and dispense 1–5 mg quantities of more than 250 different plant tissue samples in 16 h. The other steps in the processing (pretreatment, enzyme digestion, and sugar analysis) have also been largely automated and require 36 h. The process is adaptable to diverse acidic and basic, low-temperature pretreatments. Total throughput of the HTDP is 972 independent biomass samples per week. Validation of the platform was performed on brown midrib mutants of maize, which are known to have enhanced digestibility. Additional validation was performed by screening approximately 1,200 Arabidopsis mutant lines with T-DNA insertions in genes known or suspected to be involved in cell wall biosynthesis. Several lines showed highly significant (p

The in vitro fermentability of three novel dietary fibers (DFs) prepared from mushroom sclerotia, namely, Pleurotus tuber-regium, Polyporous rhinocerus, and Wolfiporia cocos, was investigated and compared with that of the cellulose control. All DF samples (0.5 g each) were fermented in vitro with a human fecal homogenate (10 mL) in a batch system (total volume, 50 mL) under strictly anaerobic conditions (using oxygen reducing enzyme and under argon atmosphere) at 37°C for 24 h. All three novel sclerotial DFs exhibited notably higher dry matter disappearance (P. tuber-regium, 8.56%; P. rhinocerus, 13.5%; and W. cocos, 53.4%) and organic matter disappearance (P. tuber-regium, 9.82%; P. rhinocerus, 14.6%; and W. cocos, 57.4%) when compared with those of the cellulose control. Nevertheless, only the W. cocos DF was remarkably degraded to produce considerable amounts of total short chain fatty acids (SCFAs) (5.23 mmol/g DF on organic matter basis, with a relatively higher molar ratio of propionate) that lowered the pH of its nonfermented residue to a slightly acidic level (5.89). Variations on the in vitro fermentability among the three sclerotial DFs might mainly be attributed to their different amounts of interwoven hyphae present (different amounts of enzyme inaccessible cell wall components) as well as the possible different structural arrangement (linkage and degree of branching) of their β-glucans.

Opuntia ficus-indica cladodes represent the green stem of the plant and are generally used as animal feed or disposed of in landfills. The present work investigated the anatomical and chemical composition of Opuntia cladodes, which form the basis of their pharmacological effects. Glucose and galacturonic acid were the main sugars of Opuntia cladodes, whereas high-performance liquid chromatography (HPLC) analysis showed the presence of mainly kaempherol and isorhamnetin glycosides (glucoside and rhamnoside). The presence of high amounts of calcium oxalate crystals was demonstrated by light microscopy on fresh and lyophilized cladodes. No antimicrobial activity was observed even after enzymatic treatment. O. ficus-indica cladodes may retain material tightly associated with cell-wall components, and this property will have the potential to greatly reduce the bioavailability of bioactive compounds.

Insoluble fibres were isolated from the two varieties of foxtail millet (white and yellow) grains and evaluated for their hypoglycaemic effects by in vitro studies. The hypoglycaemic effects of these fibres were compared with those of commercial soy insoluble fibre. The results revealed that minimum and maximum amounts of glucose were adsorbed on each sample at 10 and 200 μmol g-1 glucose concentrations respectively, indicating that the glucose adsorption capacity (GAC) of the fibre materials was proportional to glucose concentration for all samples. There was significant (P< 0.05) difference among all the fibre materials in relation to their GAC values. In the case of the effects of the fibres on glucose diffusion, the millets' insoluble fibres performed better than that of the commercial soy insoluble fibres. The glucose dialysis retardation indexes at the end of the maximum dialysis time were 1.1%, 27.4% and 22.6% for soy bean insoluble fibre, white foxtail millet insoluble fibre and yellow foxtail millet insoluble fibre in that order. The study showed that hypoglycaemic effects of yellow and white foxtail millet fibres were comparable to the commercial soy insoluble fibre.

In vitro hypoglycemic effects of different insoluble fiber-rich fractions prepared from the peel of Citrus sinensis L. cv. Liucheng

Insoluble fiber-rich fractions (FRFs), including insoluble dietary fiber, alcohol-insoluble solid, and water-insoluble solid, were isolated from the peel of Citrus sinensis L. cv. Liucheng. We found that these three FRFs could effectively adsorb glucose, retard glucose diffusion, and inhibit the activity of α-amylase to different extents. These mechanisms might create a concerted benefit in decreasing the rate of glucose absorption and eventually lower the concentration of postprandial serum glucose. The potential hypoglycemic effects of these FRFs suggested that they could be incorporated as low-calorie bulk ingredients in high-fiber foods to reduce calorie level and control blood glucose level.

Structure and digestibility of endosperm water-soluble α-glucans from different sugary maize mutants.

The structure and digestibility of endosperm water-soluble α-glucans from different sugary-1 maize mutants (Zhongtian 8#, Zhongtian 2# and Pintian 8#) were investigated. The yield of pure glucan was in the range of 25.91–34.38%. The α-glucan belonged to a typical native nano-scale particle and the average particle size was in the following order: Zhongtian 8# >Pintian 8# >Zhongtian 2#. The weight-average molar mass of glucans ranged from 1.69 to 2.08 × 107 g/mol. The branch densities and α-1,6 linkages of Zhongtian 8#, Zhongtian 2# and Pintian 8#, were 8.60%, 8.77% and 9.51%, 7.71%, 6.58% and 6.81%, respectively. The resistant starch (10.06%) of Pintian 8# was lower than other two cultivars. The study showed that water-soluble glucan exhibited α-1,4-linked backbone with α-1,6 branch sites and digestibility was influenced by granule size, ratio of α-1,4 to α-1,6 linkages, molecular fine structure in this set of sugary maize mutants.

ADP-glucose pyrophosphorylase (AGPase) regulates starch biosynthesis in higher plants and microalgae. This study measured the effect of the bacterium Azospirillum brasilense on AGPase activity in the freshwater microalga Chlorella vulgaris and formation of starch. This was done by immobilizing both microorganisms in alginate beads, either replete with or deprived of nitrogen or phosphorus and all under heterotrophic conditions, using D-glucose or Na-acetate as the carbon source. AGPase activity during the first 72 h of incubation was higher in C. vulgaris when immobilized with A. brasilense. This happened simultaneously with higher starch accumulation and higher carbon uptake by the microalgae. Either carbon source had similar effects on enzyme activity and starch accumulation. Starvation either by N or P had the same pattern on AGPase activity and starch accumulation. Under replete conditions, the population of C. vulgaris immobilized alone was higher than when immobilized together, but under starvation conditions A. brasilense induced a larger population of C. vulgaris. In summary, adding A. brasilense enhanced AGPase activity, starch formation, and mitigation of stress in C. vulgaris.

Effects of gamma irradiation on starch digestibility of rice with different resistant starch content.

Three rice cultivars (RS3M, RS4H and RS5L) differing in resistant starch contents but similar in genetic background were chosen to study the effects of gamma irradiation on starch physicochemical properties and digestibility. Irradiation increases the resistant starch content in all the three cultivars and in a dose-dependent manner in rice with low-resistant starch content (RS5L). Irradiation decreases apparent amylose content and gelatinisation temperature and changed the starch granule structure, while increasing V-type crystallinity. Starch enzymatic hydrolysis rate was reduced following irradiation, and the effect of irradiation on reducing starch digestibility was negatively correlated with resistant starch content. Treatment with gamma irradiation has therefore a potential for increasing resistant starch content and producing low digestibility of starch in common rice.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.

Send the results of the kit standard, blank samples and the results obtained for your sample, in the relevant MegaCalc spreadsheet (if available) to Megazyme (cs@megazyme.com). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.

State the kit lot number being used (this is found on the outside of the kit box).

State which assay format was used (refer to the relevant page in the kit booklet if necessary).

State exact details of any modifications to the standard procedure that is provided by Megazyme.

State the sample type and describe the sample preparation steps if applicable.

The test is best run at pH 7.4. The reagent is buffered at this pH. Using different pH values will affect results, i.e. it may take longer to reach this end point; but the same end-point value should be obtained if not too far away from this pH value.

The test is set up to measure between 10 and 100 micrograms per assay (i.e. 0.1 mL of 0.1 to 1.0 mg/mL). You may prefer to use 3.0 mL of GOPOD reagent mixture plus 1.0 mL of sample; in this case the concentration range in the material being analysed (diluted) should be ~10 to 100 micrograms per mL. The test is linear up to an absorbance of 1.4 (final assay volume of 3.2 mL). If the final volume is 4.0 mL, then linearity will be up to about 1.0 absorbance units.

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Sometimes the addition of the last assay component can cause a small negative absorbance change in the blank samples due to a dilution effect and in such cases it is recommended that the real absorbance values be used in the calculation of results.